This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-CA-103 Synthetic Biology. In this proposal, the Shen and Califano laboratories will combine their complementary expertise in stem cell biology and computational systems biology to pursue an integrated, systems-level analysis of the molecular basis of stem cell pluripotency and self-renewal. In particular, these studies will use a systems biology/reverse engineering approach to infer molecular interaction networks, or interactomes, that mediate stem cell properties. The resulting interactomes will be highly relevant for the elucidation of normal developmental mechanisms and their alteration in cancer. Despite rapid advances in recent years, the Master Regulator (MR) genes controlling pluripotency, self renewal, and lineage-specific differentiation in embryonic stem cells (ESC) and other pluripotent stem cells have only been partially characterized. Although many studies have focused on mouse and human ESC, other stem cell lines such as mouse Epiblast Stem Cells (EpiSC) also display pluripotency and unlimited selfrenewal. Notably, mouse EpiSC appear to be more similar to human ESC than are mouse ESC. Consequently, dissection of the interactome governing mouse EpiSC pluripotency and self-renewal should provide key insights into cross-species similarities and differences with human ESC, and would allow rational experimental testing in embryos to validate properties of pluripotency in vivo. These studies would also elucidate mechanisms underlying reprogramming of somatic cell types into induced Pluripotent Stem Cells (iPSC), and would help define the connection between pluripotency and tumorigenesis. Thus, we propose using mouse EpiSC, human ESC, and human iPSC lines for the inference and comparative analysis of interactomes that govern pluripotency and self-renewal. Importantly, the proposed approach is unbiased and will allow both the recapitulation of known mechanisms in a context-dependent fashion, as well as systematic elucidation of novel MR genes associated with these processes for experimental validation. We therefore propose the following specific aims: 1) Dissection of the regulatory networks that govern pluripotency and self-renewal by applying genome-wide reverse-engineering algorithms to generate Stem Cell Interactomes (SCIs) for mouse EpiSC and human ESC. 2) Identification of key regulatory genes for pluripotency and self-renewal by interrogation of the SCIs for mouse (EpiSC) and human (ESC) using a novel Master Regulator Analysis (MRA) algorithm. In addition, the human ESC interactome will be interrogated using differentiation signatures for human iPSC to understand regulatory differences and MR gene overlap. 3) Functional validation of candidate regulatory genes for pluripotency and self-renewal by overexpression and knock-down methods in experimental assays for pluripotency, self-renewal, and lineage-specific differentiation. PUBLIC HEALTH RELEVANCE: Our proposed studies will investigate central biological mechanisms for stem cell self-renewal, pluri/multipotency, and lineage-specific differentiation that are likely to be conserved in multiple biological contexts, including in cancer and specifically in putative cancer stem cells. In principle, our work should establish a rational basis for the experimental manipulation of stem cell differentiation as well as reprogramming of somatic cell types into induced Pluripotent Stem Cells (iPSC). Thus, this collaboration between stem cell and systems biologists represents an initial step toward the assembly of a synthetic biological platform for modeling stem cell processes in regenerative medicine and cancer.